Transposon Defense by Endo-siRNAs,piRNAs and Somatic pilRNAs in Drosophila: Contributions of Loqs-PD and R2D2

Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. We provide further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway.     

The PIWI‐Interacting RNA Molecular Pathway: Insights From Cultured Silkworm Germline Cells

The PIWI‐interacting RNA (piRNA) pathway, one of the major eukaryotic small RNA silencing pathways, is a genome surveillance system that silences selfish genes in animal gonads. piRNAs guide PIWI protein to target genes through Watson–Crick RNA–RNA base‐parings. Loss of piRNA function causes genome instability, inducing failure in gametogenesis and infertility. Studies using fruit flies and mice as key experimental models have resulted in tremendous progress in understanding the mechanism underlying the piRNA pathway. Recent work using cultured silkworm germline cells has also expanded our knowledge of piRNA biogenesis in particular, since these silkworm cells are the only cells of germline origin that can be cultured. In this review, we describe elucidation of the piRNA pathway using cultured silkworm cells as an experimental model by focusing on recent work in biochemistry and structural biology. Earlier studies that made important contributions to the field are also described.     

Small noncoding RNAs in the germline

Small noncoding RNAs have emerged as potent regulators of gene expression, especially in the germline. We review the biogenesis and regulatory function of three major small noncoding RNA pathways in the germline: The small interfering RNA (siRNA) pathway that leads to the degradation of target mRNAs, the microRNA (miRNA) pathway that mostly represses the translation of target mRNAs, and the newly discovered Piwi-interacting RNA (piRNA) pathway that appears to have diverse functions in epigenetic programming, transposon silencing, and the regulation of mRNA translation and stability. The siRNA and miRNA pathways are present in the germline as well as many somatic tissues, whereas the piRNA pathway is predominantly confined to the germline. Investigation of the three small RNA pathways has started to reveal a new dimension of gene regulation with defining roles in germline specification and development.     

Cloning, characterization and widespread expression analysis of testicular piRNA-like chicken RNAs

Piwi-interacting RNAs (piRNAs) are small RNAs abundant in the germline that have been implicated in germline development and maintenance of genomic integrity across several animal species including human, mouse, rat, zebrafish and drosophila. Tens of thousands of piRNAs have been discovered, yet abundant piRNAs have still not been detected in various eukaryotic organisms. This is a report on the characterization, cloning and expression profiling of piRNA-like chicken RNAs. Here, we identified 19 piRNAs, each 23–39 nucleotides long, from chicken testis using a small RNA cDNA library and T-A cloning methods. Three different pilRNAs were selected according to size, homology and secondary structure for temporal and spatial expression by Q-PCR technology in different tissues at five growth and four development stages of Chinese indigenous Rugao chickens (RG) and introduced recessive white feather chickens (RW). We found that, consistent to other organisms, pilRNA-encoding sequences within the chicken genome were asymmetrically distributed on the chromosomes while displaying a preference for intergenic regions across the genome. Interestingly, unlike miRNAs with unique stem-loop structures (mature miRNAs form stem section and the rest form loop section), distinct secondary structures of pilRNAs were predicted. In addition, chicken pilRNAs were not only abundant in the germline but also existed in somatic tissues, where, expression levels were influenced mainly by different pilRNAs, breed and gender. Taken together, our results suggest that two distinct secondary structures exist between pilRNAs and miRNAs, which may clarify the splicing and processing mechanisms of the two small RNAs are possible different. Moreover, our results suggest that pilRNAs may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. Additionally, different pilRNAs may be involved in the unique regulatory machinery of complex biological processes.     

Bombyx small RNAs: genomic defense system against transposons in the silkworm, Bombyx mori

Selfish genetic elements called transposons can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Expansion of transposons can occur when novel transposition events are transmitted to subsequent generations after germline hopping. Therefore, organisms seem likely to have evolved defense mechanisms to silence transposons in the germline. Recently, small RNAs interacting with Piwi proteins (piwi-interacting RNAs: piRNAs) have been demonstrated to be involved in genomic defense mechanism against transposons. Here, we show that piRNA-like small RNAs are present abundantly in the Bombyx ovary. We cloned 38,493 kinds of Bombyx small RNA from the ovary and performed functional characterization. Bombyx small RNAs showed a unimodal length distribution with a peak at 28nt and a strong bias for U at the 5' end. We found that 12,869 kinds of Bombyx small RNAs were associated with transposons or repetitive sequences. We classified them as repeat-associated small interfering RNAs (rasiRNAs), a subclass of piRNAs. Notably, antisense rasiRNAs have a strong bias toward U at 5' ends; in contrast, sense rasiRNAs have a strong bias toward A at nucleotide position 10, indicating that the piRNA amplification loop proposed in Drosophila is evolutionarily conserved in Bombyx. These results suggest that Bombyx small RNAs regulate transposon activity.     

Lesser known relatives of miRNA

Since the discovery of RNAi (RNA interference) major attention has focused on studying miRNA (microRNA) and siRNA (small interfering RNA). However, within the last few years, several other small ncRNAs (non-coding RNAs) have been discovered and thus various newer acronyms representing these ‘other’ classes of small ncRNAs have populated the literature. Of these, piRNA (Piwi-interacting RNA) has been gaining importance because of its role as the guardian of the germline genome. Some of the other newly discovered small ncRNAs have been mostly reported in plants, and they are yet to be studied more comprehensively. Nevertheless, piRNA and the ‘other’ small ncRNAs deserve some discussion because they are members of the increasingly large family of small ncRNAs.     

Identification and Characterization of piRNA-Like Small RNAs in the Gonad of Sea Urchin (Strongylocentrotus nudus)

Piwi-interacting RNAs (piRNAs) and their partner PIWI proteins play an essential role in fertility, germline stem cell development, as well as the basic control and evolution of animal genomes. However, research was rare with regard to piRNA population in sea urchin, a model animal intensively used for development and genetics studies. Utilizing Solexa sequencing, we present an identification of 13,051 piRNA-like RNAs expressed in male gonad of Strongylocentrotus nudus. Out of 202 tested RNAs, 94 sequences were confirmed to express in female gonad using microarray assay, suggesting that both male and female gonads are piRNA-like RNA-enriched organs. These RNAs with "U" at the 5' end or "A" at position of 10, in size from 26 to 30 nucleotides, were predominantly 28?nt in length and tend to be clustered in small regions in genome, achieving the longest piRNA-like RNA-enriched region about 5.5?kb in scaffold78427. Alignment results showed 11 RNAs were homologous to the known piRNAs. Furthermore, BLASTn searching against sea urchin repeat element database showed these piRNA-like RNAs matched to 101 types of DNA transposons and retrotransposons, of which SPRP1, Harbinger-N2, piggyBac-N10, SINE2-1, and piggyBac-N11 were the most frequent hit elements, suggesting a transposon silencing function of these piRNA-like RNAs.     

piRNA和PIWI蛋白的功能机制研究进展

piRNA是2006年7月在动物生殖细胞中发现的一类新小分子非编码RNA。piRNA特异地与PIWI家族蛋白相互作用,因此,被命名为PIWI-interacting RNA,简称piRNA。这类长度在26～32核苷酸的小分子非编码RNA代表了一个生殖细胞转座子沉默的独特小RNA通路。它们可能通过与PIWI家族蛋白质相互作用,在表观遗传学水平和转录后水平沉默转座子等基因组自私性遗传元件,参与生殖干细胞自我维持和分化命运决定、减数分裂、精子形成等生殖相关事件。在piRNA发现后短短数年的时间,对其生物发生、功能及作用机制的研究都取得了诸多重大突破。该文就piRNA研究的最新研究进展作一简述。     

piRNA analysis framework from small RNA-Seq data by a novel cluster prediction tool - PILFER

With the increasing number of studies focusing on PIWI-interacting RNA (piRNAs), it is now pertinent to develop efficient tools dedicated towards piRNA analysis. We have developed a novel cluster prediction tool called PILFER (PIrna cLuster FindER), which can accurately predict piRNA clusters from small RNA sequencing data. PILFER is an open source, easy to use tool, and can be executed even on a personal computer with minimum resources. It uses a sliding-window mechanism by integrating the expression of the reads along with the spatial information to predict the piRNA clusters. We have additionally defined a piRNA analysis pipeline incorporating PILFER to detect and annotate piRNAs and their clusters from raw small RNA sequencing data and implemented it on publicly available data from healthy germline and somatic tissues. We compared PILFER with other existing piRNA cluster prediction tools and found it to be statistically more accurate and superior in many aspects such as the robustness of PILFER clusters is higher and memory efficiency is more. Overall, PILFER provides a fast and accurate solution to piRNA cluster prediction.     

GPAT2, a mitochondrial outer membrane protein,in piRNA biogenesis in germline stem cells

piRNA (PIWI-interacting RNA) is a germ cell–specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.     

Identification of piRNAs in the central nervous system

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs generated by a conserved pathway. Their most widely studied function involves restricting transposable elements, particularly in the germline, where piRNAs are highly abundant. Increasingly, another set of piRNAs derived from intergenic regions appears to have a role in the regulation of mRNA from early embryos and gonads. We report a more widespread expression of a limited set of piRNAs and particularly focus on their expression in the hippocampus. Deep sequencing of extracted RNA from the mouse hippocampus revealed a set of small RNAs in the size range of piRNAs. These were confirmed by their presence in the piRNA database as well as coimmunoprecipitation with MIWI. Their expression was validated by Northern blot and in situ hybridization in cultured hippocampal neurons, where signal from one piRNA extended to the dendritic compartment. Antisense suppression of this piRNA suggested a role in spine morphogenesis. Possible targets include genes, which control spine shape by a distinctive mechanism in comparison to microRNAs.     

piRNA的功能研究进展

piRNA是一类种类繁多的小RNA,通常在生殖类细胞中表达,其功能是抑制转座子的转座,维持基因组结构的稳定性。对线虫piRNA研究发现,piRNA还具有记忆基因表达的功能。体细胞和癌细胞中piRNA的发现,更凸显了piRNA功能的重要性和多样性。本文梳理了近几年来piRNA功能研究的最新成果,包括piRNA在调控转座子、mRNA、lncRNA、DNA甲基化修饰、染色体表观修饰等方面的功能,同时也探讨了piRNA和癌症的关系。     

piRNA/PIWI功能调控与精子发生

piRNAs(PIWI-interacting RNAs)是一类与PIWI相互作用的小非编码RNAs(small noncoding RNAs, sncRNAs),其长度介于24~32 nt,特异性地在动物生殖腺细胞中表达。近来研究表明piRNA/PIWI系统在动物生殖腺细胞的基因组转座元件沉默及转录后调控mRNAs方面具有重要功能。最近,中国科学院上海生物化学与细胞生物学研究所刘默芳课题组的一项研究表明,在人和小鼠的精子发生过程中,PIWI (鼠源同源蛋白MIWI、人源同源蛋白HIWI)的严格代谢调控至关重要。以此为契机,本文综述了piRNA/PIWI在哺乳动物(主要是小鼠和人)精子发生过程中调控功能的研究进展。